p47v‐crk (v‐Crk), a transforming gene product containing Src homology (SH)‐2 and ‐3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, a 125‐135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v‐Crk, suggesting a direct signal mediator of v‐Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3‐containing signaling molecule with a cluster of multiple putative SH2‐binding motifs of v‐Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60v‐src (v‐Src), as well as by v‐Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v‐Src‐associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk‐associated substrate) is a common cellular target of phosphorylation signal via v‐Crk and v‐Src oncoproteins, and its unique structure indicates the possible role of p130Cas in assembling signals from multiple SH2‐containing molecules.

Introductory paragraph The adoptive transfer of anti-CD19 chimeric antigen receptor (CAR)-engineered T cells has shown impressive clinical responses in patients with refractory B-cell malignancies1–7. However, the therapeutic effects of CAR-T cells targeting other malignancies have not yet resulted in significant clinical benefit8–11. Although inefficient tumor trafficking and various immunosuppressive mechanisms can impede CAR-T cell effector responses, the signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals, including T cell receptor (TCR) engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3)12. However, CAR gene constructs currently being tested in the clinic contain a CD3z (TCR signaling) domain and a costimulatory domain(s) but not a domain transmitting signal 313–18. Here, we have developed a novel CAR construct capable of inducing cytokine signaling upon antigen stimulation. This new generation CD19 CAR encodes a truncated cytoplasmic domain of IL-2Rβ and a STAT3-binding YXXQ motif together with CD3z and CD28 domains (28-ΔIL2RB-z (YXXQ)). The 28-ΔIL2RB-z (YXXQ) CAR-T cells showed antigen-dependent JAK-STAT3/5 pathway activation, which promoted their proliferation and prevented terminal differentiation in vitro. The 28-ΔIL2RB-z (YXXQ) CAR-T cells demonstrated superior in vivo persistence and antitumor effects in both liquid and solid tumor models compared with CAR-T cells with a CD28 or 4-1BB domain alone. Taken together, these results suggest that our new generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicities in the clinic. Clinical translation of this novel CAR is warranted.

During a search for genes that maintain T cell quiescence, we determined that Tob, a member of an anti-proliferative gene family, was highly expressed in anergic T cell clones. Tob was also expressed in unstimulated peripheral blood T lymphocytes and down-regulated during activation. Forced expression of Tob inhibited T cell proliferation and transcription of cytokines and cyclins. In contrast, suppression of Tob with an antisense oligonucleotide augmented CD3-mediated responses and abrogated the requirement of costimulation for maximal proliferation and cytokine secretion. Tob associated with Smad2 and Smad4 and enhanced Smad DNA-binding. The inhibitory effect of Tob on interleukin 2 (IL-2) transcription was not mediated by blockade of NFAT, AP-1 or NF-kappaB transactivation but by enhancement of Smad binding on the -105 negative regulatory element of the IL-2 promoter. Thus, T cell quiescence is an actively maintained phenotype that must be suppressed for T cell activation to occur.